PROJECT SUMMARY The central goal of our proposed research is to obtain a functional understanding of the native and toxin-exposed gut microbiome in xenometabolism from phylum to taxa to specific enzymes. The genomes of the microorganisms within the gut microbiome encode an extensive capacity for the metabolism of dietary compounds, pharmaceutical drugs, environmental chemicals, persistent organic pollutants, and other xenobiotics. Gut microbiota-xenobiotic interactions are also reciprocal, as exposure to xenobiotics can perturb the metabolic function of the microbiome. Our current understanding of microbiome functions are largely inferred from corollary measurements that can ascribe the potential for a function, but can not actually confirm that the function is present and active. We will make a significant advance in the understanding of microbial metabolic functions and how they influence human health by applying an activity-based protein profiling (ABPP) platform for in situ analysis of taxa and enzyme functional activities. By being able to identify the functionally active cells and enzymes within the microbiome, meaningful conclusions can be drawn about the connection between the gut microbiome and xenobiotic metabolism. Our ABPP platform will deploy chemical probes that target only catalytically active enzymes involved in xenometabolism in the gut microcbiome. The probes target the functional enzymes, but will enable isolation, characterization, and quantification of both the cells expressing those functions, and the specific enzymes catalyzing xenobiotic metabolism. With our ABPP platform we will address the central hypothesis that xenometabolism in the gut microbiome can be performed with phylogenetically disparate populations and different enzymes. We will initially develop a suite of new activity-based probes for five central xenobiotic metabolizing enzyme families in the gut microbiome. We will apply these to mouse gut and fecal microbiomes, and use flow cytometry, imaging, and proteomics to characterize functional cells and enzymes. We will also evaluate how exposure to a toxin, which is also a strong agonist of the aryl hydrocarbon receptor, impacts the compositional and functional environment of the microbiome. Completion of this project will provide a rich new functional understanding of microbial xenobiotic metabolism at the level of specific cells and enzymes. Our research will also yield a platform for future microbiome studies to evaluate individual variability and susceptibility to diseases, to understand consequences associated with xenobiotic exposures in adults and developing children, as well as for the future of precision medicine.